Health Council of the Netherlands Phenacetin Evaluation of the carcinogenicity and genotoxicity Gezondheidsraad Health Council of the Netherlands Aan de staatssecretaris van Sociale Zaken en Werkgelegenheid Onderwerp Uw kenmerk Ons kenmerk Bijlagen Datum : aanbieding advies Phenacetin : DGV/MBO/U-932342 : U-7412/BvdV/fs/246-C17 :1 : 13 november 2012 Geachte staatssecretaris, Graag bied ik u hierbij het advies aan over de gevolgen van beroepsmatige blootstelling aan fenacetine. Dit advies maakt deel uit van een uitgebreide reeks waarin kankerverwekkende stoffen worden geclassificeerd volgens richtlijnen van de Europese Unie. Het gaat om stoffen waaraan mensen tijdens de beroepsmatige uitoefening kunnen worden blootgesteld. Dit advies is opgesteld door een vaste subcommissie van de Commissie Gezondheid en beroepsmatige blootstelling aan stoffen (GBBS), de Subcommissie Classificatie van carcinogene stoffen. Het advies is getoetst door de Beraadsgroep Gezondheid en omgeving van de Gezondheidsraad. Ik heb het advies vandaag ter kennisname toegezonden aan de staatssecretaris van Infrastructuur en Milieu en aan de minister van Volksgezondheid, Welzijn en Sport. Met vriendelijke groet, prof. dr. W.A. van Gool, voorzitter Bezoekadres Postadres Parnassusplein 5 Postbus 16052 2 5 11 V X D e n 2500 BB Den Haag E - m a i l : b . v. d . v o e t @ g r. n l Te l e f o o n ( 0 7 0 ) 3 4 0 7 4 4 7 w w w. g r. n l Haag Phenacetin Evaluation of the carcinogenicity and genotoxicity Subcommittee on the Classification of Carcinogenic Substances of the Dutch Expert Committee on Occupational Safety, a Committee of the Health Council of the Netherlands to: the State Secretary of Social Affairs and Employment No. 2012/21, The Hague, November 13, 2012 The Health Council of the Netherlands, established in 1902, is an independent scientific advisory body. Its remit is “to advise the government and Parliament on the current level of knowledge with respect to public health issues and health (services) research...” (Section 22, Health Act). The Health Council receives most requests for advice from the Ministers of Health, Welfare & Sport, Infrastructure & the Environment, Social Affairs & Employment, Economic Affairs, Agriculture & Innovation, and Education, Culture & Science. The Council can publish advisory reports on its own initiative. It usually does this in order to ask attention for developments or trends that are thought to be relevant to government policy. Most Health Council reports are prepared by multidisciplinary committees of Dutch or, sometimes, foreign experts, appointed in a personal capacity. The reports are available to the public. The Health Council of the Netherlands is a member of the European Science Advisory Network for Health (EuSANH), a network of science advisory bodies in Europe. The Health Council of the Netherlands is a member of the International Network of Agencies for Health Technology Assessment (INAHTA), an international collaboration of organisations engaged with health technology assessment. I NA HTA This report can be downloaded from www.healthcouncil.nl. Preferred citation: Health Council of the Netherlands. Phenacetin. Evaluation of the carcinogenicity and genotoxicity. The Hague: Health Council of the Netherlands, 2012; publication no. 2012/21. all rights reserved ISBN: 978-90-5549-920-5 Contents Samenvatting 9 Executive summary 11 1 1.1 1.2 1.3 Scope 13 Background 13 Committee and procedures 13 Data 14 2 2.1 2.2 General information 15 Identity and physicochemical properties 15 IARC classification 16 3 3.1 3.2 Carcinogenicity 17 Observations in humans 17 Carcinogenicity studies in animals 22 4 4.1 Mode of action 27 Genotoxic mode of action 27 Contents 7 5 5.1 5.2 Classification 31 Evaluation of data on carcinogenicity and genotoxicity 31 Recommendation for classification 32 References 33 A B C D E F G H I Annexes 39 Request for advice 41 The Committee 43 The submission letter 45 Comments on the public review draft 47 IARC Monograph 49 Human data 51 Animal data 57 Genotoxicity data 59 Carcinogenic classification of substances by the Committee 61 8 Phenacetin Samenvatting Op verzoek van de minister van Sociale Zaken en Werkgelegenheid evalueert en beoordeelt de Gezondheidsraad de kankerverwekkende eigenschappen van stoffen waaraan mensen tijdens het uitoefenen van hun beroep kunnen worden blootgesteld. De evaluatie en beoordeling worden verricht door de subcommissie Classificatie van Carcinogene Stoffen van de Commissie Gezondheid en Beroepsmatige Blootstelling aan Stoffen van de raad, hierna kortweg aangeduid als de commissie. In het voorliggende rapport neemt de Commissie fenacetine onder de loep. Fenacetine werd vanaf 1887 tot ongeveer 1980 gebruikt als pijnstiller. Omdat er steeds meer aanwijzingen kwamen dat chronisch gebruik van fenacetine vormen van nierproblemen kan veroorzaken, is de stof niet meer als geneesmiddel geregistreerd. Fenacetine wordt vaak versneden aangetroffen in illegaal verkrijgbare cocaïne. Op basis van de beschikbare gegevens leidt de commissie af dat fenacetine kankerverwekkend is voor de mens. Zij beveelt aan om de stof te classificeren in categorie 1A.* De commissie concludeert verder dat de stof een stochastisch genotoxisch werkingsmechanisme heeft. * Volgens het classificatiesysteem van de Gezondheidsraad (zie bijlage I). Samenvatting 9 10 Phenacetin Executive summary At request of the Minister of Social Affairs and Employment, the Health Council of the Netherlands evaluates and judges the carcinogenic properties of substances to which workers are occupationally exposed. The Evaluation is performed by the subcommittee on the Classification of Carcinogenic Substances of the Dutch Expert Committee on Occupational Standards of the Health Council, hereafter called the Committee. In this report, the Committee evaluated phenacetin. Phenacetin was after the introduction in 1887 up to the early 1980s used as an analgesic drug. Because chronic use of phenacetin is suspected to cause renal problems the registration of the drug has been discontinued. Phenacetin is being used as a cutting agent to adulterate illegally supplied cocaïne. Based on the available information, the Committee is of the opinion that phenacetin is carcinogenic to humans and recommends to classify the substance in category 1A.* The Committee is furthermore of the opinion that phenacetin acts by a stochastic genotoxic mechanism. * According to the classification system of the Health Council (see Annex I). Executive summary 11 12 Phenacetin Chapter 1.1 1 Scope Background In the Netherlands a special policy is in force with respect to occupational use and exposure to carcinogenic substances. Regarding this policy, the Minister of Social Affairs and Employment has asked the Health Council of the Netherlands to evaluate the carcinogenic properties of substances, and to propose a classification (see Annex A). In addition to classifying substances, the Health Council also assesses the genotoxic properties of the substance in question. The assessment and the proposal for a classification are expressed in the form of standard sentences (see Annex I). This report contains the evaluation of the carcinogenicity of phenacetin 1.2 Committee and procedures The evaluation is performed by the subcommittee on the Classification of Carcinogenic Substances of the Dutch Expert Committee on Occupational Standards of the Health Council, hereafter called the Committee. The members of the Committee are listed in Annex B. The submission letter (in English) to the State Secretary can be found in Annex C. In June 2012, the President of the Health Council released a draft of the report for public review. No comments were received on the draft document. Scope 13 1.3 Data The evaluation and recommendation of the Committee is based on scientific data, which are publicly available. The starting points of the Committees’ reports are, if possible, the monographs of the International Agency for Research on Cancer (IARC). This means that the original sources of the studies, which are mentioned in the IARC-monograph, are reviewed only by the Committee when these are considered most relevant in assessing the carcinogenicity and genotoxicity of the substance in question. The evaluation of the carcinogenicity of phenacetin has been based on IARC evaluations (IARC volume 13 (1977), IARC volume 24 (1980), IARC supplement 7 (1987) and IARC volume 100A (2011))1-4 (in Annex E a summary is given of the IARC data) and additional scientific data, which are publicly available. Additional data were obtained from the online databases Toxline, Medline and Chemical Abstracts covering the period 1978 to September 2012 using phenacetin and CAS no 62-44-2 as key words in combination with key words representative for carcinogenesis and mutagenesis. The new relevant data were included in this report. 14 Phenacetin Chapter 2.1 2 General information Identity and physicochemical properties Chemical name CAS registry number EINECS-number EEC-number RTECS-number Synonyms Appearance : : : : : : : Occurrence Use : : Molecular formula Structural formula : : Molecular weight Boiling point Melting point Vapour pressure Vapour density (air = 1) Solubility Stability and reactivity EU Classification : : : : : : : : General information N-(4-ethoxyphenyl)acetamide1 62-44-2 200-533-05 N-(4-ethoxyphenyl), acetyl-phenetidine, 1-acetamido-4-ethoxybenzene odorless, white, glistening crystals, usally scales or as fine white, crystalline powder6 analgesic and antipyretic drug in human and veterinary medicine.2; registration in the Netherlands was discontinued in 1984 because of serious side effects on the kidney; illegal use as adulterant in cocaine powder C10-H13-N-O26 179.226 242-245°C6 134-135°C6 Slightly soluble in water (1 in 1,300)2 Unstable to oxidizing agents, iodine and nitrating agents2 Not classified in Annex I of Directive 67/548/EEC 15 2.2 IARC classification In 2011, IARC concluded : There is sufficient evidence in humans for the carcinogenicity of analgesic mixtures containing phenacetin. Analgesic mixtures containing phenacetin cause cancer of the renal pelvis, and of the ureter. There is limited evidence in experimental animals for the carcinogenicity of analgesic mixtures containing phenacetin. There is sufficient evidence in humans for the carcinogenicity of phenacetin. Phenacetin causes cancer of the renal pelvis, and of the ureter. There is sufficient evidence in experimental animals for the carcinogenicity of phenacetin. Analgesic mixtures containing phenacetin are carcinogenic to humans (Group 1). Phenacetin is carcinogenic to humans (Group 1). 16 Phenacetin Chapter 3.1 3 Carcinogenicity Observations in humans Many case report studies showed the existence of renal pelvic and other urothelial tumours in patients who have used large amounts of phenacetincontaining analgesics.7-16, 17-22 A vast amount of case-control studies23-28, 29-44 have been published. These studies show that phenacetin-containing analgesics are part of the etiology of renal pelvic, urothelial and bladder cancer. Most of the exposed individuals in these case-control studies are exposed to phenacetin-containing analgesics, which makes it difficult to investigate the effect of exposure to phenacetin only. Most of the studies were published 15-20 years ago, due to the fact that phenacetin-containing products had been off the market in most countries for decades now. Recent studies were not published because the lack of long-time phenacetin users. The case-control studies have been summarized in the following paragraphs and in Annex F. Renal pelvis cancer McCredie et al. (1986) conducted a hospital based case-control study in New South Wales, Australia to investigate the risk factors for renal cancer. Sixty six cases of renal pelvis cancer, 86 cases of renal parenchyma cancer and 751 controls were collected between 1970 and 1982 in Sidney, Australia. Information Carcinogenicity 17 on consumption of phenacetin-containing analgesics was obtained through completion of a structured questionnaire at interview. Pathologists classified the tumours according to their histological appearances and sought evidence of ‘intermediate’ or ‘advanced’ renal papillary necrosis (RPN). Cases were excluded if the presence or absence of RPN could not be established. RPN and regular consumption of phenacetin both increased the risk for renal pelvis cancer. The risk of renal pelvis cancer increased nearly 4 times for regular consumers of phenacetin without RPN (RR: 3.6, 95% CI: 1.6-8.1) and 20 times for regular consumers of phenacetin with RPN (RR: 20, 95% CI: 12-34), compared to nonconsumers without RPN.36 McCredie et al. (1988) also conducted a population-based case control study in New South Wales, Australia to investigate the risk of developing renal cancer papillary necrosis and cancer of the renal pelvis, ureter or bladder associated with consumption of either phenacetin or paracetamol. Data were acquired from 381 cases (identified between 1978 and 1982) and 808 controls. The risk of cancer of the renal pelvis was statistically significantly increased nearly 6 and 8-fold with a lifetime consumption of respectively, > 0.1 kg (OR: 5.7, 95% CI: 3.2-10.0) and > 1 kg (OR: 7.9, 95% CI: 4.6-13.8) phenacetin.37 In another population-based case control study in New South Wales, Australia, McCredie et al. (1993) investigated the consumption of phenacetin and paracetamol and the risk of cancer of the kidney and renal pelvis, using data of 489 cases of renal-cell cancer and 147 cases of renal pelvic cancer diagnosed in 1989 and 1990, together with 523 controls from the electoral rolls. A doserelated increase in the risk of cancer of the renal pelvis was observed in consumers of phenacetin/aspirin compounds. When used according to the definition of “taken at least 20 times in lifetime” phenacetin/aspirin compounds increased the risk of renal pelvic cancer more than a 12-fold (RR: 12.2, 95% CI: 6.8-22.2).39 McLaughlin et al. (1985) conducted a population-based case-control study of renal cancer (495 cases of renal cell cancer, 74 cases of renal pelvis cancer and 697 controls) in Minneapolis, USA. Patients were collected in the period 19741979. Patients and the control group were interviewed in 1980 about the use of analgesic drugs. Information of different variables was obtained, including the use of analgesic drugs (phenacetin-containing, acetaminophen-containing and aspirin). A drug was considered phenacetin-containing if phenacetin was included in the formulation from 1955 to 1974. Exposures after 1973 were excluded for analysis. The groups were divided in male/female and in never, ever, irregular and regular (subdivided in ≤ 36 months and > 36 months) users. Long-term regular use of phenacetin-containing drugs was associated with an 18 Phenacetin increase in risk for renal pelvic cancer among males (OR: 8.1, 95% CI: 1.2-62), but not among females (4.2, 95% CI: 0.4-42).41 Pommer et al. (1999) conducted a case-control study in the area of the former West Berlin, including 647 new diagnosed cases of urethelial cancer (571 bladder, 25 ureter and 51 renal pelvis cancer cases) from eight hospitals of the study area between 1990 and 1995 and 647 population-based controls. Intake of more than 1 kg phenacetin in analgesic mixtures was associated with an increased risk (not significantly) of renal pelvic cancer (OR of 5.3, 95% CI: 0.3-81).43 Ureter cancer and/or renal pelvis cancer Several of the case-control studies (including two studies which are already described above by McCredie et al.,1988, Pommer et al., 199937,43) also analysed the risk of phenacetin-containing analgesics consumption for the development of ureter cancer (alone or together with renal pelvic cancer). In the populationbased case-control study in New South Wales, Australia by McCredie et al. (1988)37 no association was found between ureter cancer and a lifetime consumption of > 0.1 kg (OR: 0.7, 95% CI: 0.3-2.2) or > 1 kg phenacetin (OR: 1.2, 95% CI: 0.5-3.0). In the case-control study in the area of the former West Berlin by Pommer et al. (1999)43 no association was found between the number of renal pelvis cancer and ureter cancer combined and a lifetime intake of more than 1 kg phenacetin in analgesic mixtures (OR of 1.8, 95% CI: 0.2-13). Jensen et al. (1989)33 conducted a case-control study (96 cases and 294 controls, identified between 1979 and 1982) in Denmark to investigate the risk of analgesic intake (phenacetin and/or aspirin) and cancer of the renal pelvis and ureter. Seventy nine percent of the tumours were located in the renal pelvis (including calyces). There was an indication of a dose-effect relationship for phenacetin-containing analgesics and cancer of the renal pelvis and ureter. A statistically significant increase in relative risk (RR) was seen for female users of phenacetin-containing analgesics (RR: 4.2, 95% CI: 1.5-12.3), but not for male users (RR: 2.4, 95% CI: 0.9-6.8).33 Linet et al. (1995) investigated 502 cases (308 renal pelvis cancer and 194 ureter cancer, identified between 1983 and 1986) and 496 controls in a population-based case-control study in New Jersey, Iowa and Los Angeles, USA. Neither cumulative lifetime ingestion nor duration of regular use of phenacetin, whether alone or in combination with acetaminophen or aspirin, was associated with significantly increased risk of renal pelvis and ureter cancer. Although this Carcinogenicity 19 study contained a large amount of cases, it only contained small number of regular analgesic users.35 Renal cell cancer Three case-control studies on renal pelvis cancer, which are already described above, also analysed the risk of phenacetin-containing analgesics consumption for the development of renal cell cancer.36,41. In the population-based case-control study in Minneapolis, US of McLaughlin et al. (1985)41 (described above), long-term regular use of phenacetin-containing drugs was associated with a statistically significant increase in risk for renal cell cancer in women (OR: 1.7, 95% CI: 1.1-2.7 for ever-users and OR: 1.7, 95% CI: 1.1-2.6 for irregular-users compared to never users). In another population-based case-control study by McLaughlin et al.(1992)42 in Shanghai, China (154 cases and 157 controls) regular use of phenacetincontaining analgesics (at least 2 times a week for a period of at least 2 weeks) was not associated with renal cell cancer (OR: 2.3, 95% CI: 0.7-7.0). In the hospital based case-control study in New South Wales, Australia of McCredie et al. (1986)36 (described above), regular use of phenacetin-containing analgesics increased the risk of cancer of the renal parenchyma (RR: 2.5, 95% CI: 1.3-4.9.), but was not increased by the presence of renal papillary necrosis (RPN). Thus, unlike renal pelvis cancer, the relationship between consumption of phenacetin-containing analgesics and renal parenchyma appears to be a direct one without any intervening effect of RPN. In the population-based case-control study in New South Wales, Australia by McCredie et al. (1993) (described above), no association was found between the number of renal-cell cancers and consumption of phenacetin/aspirin compounds (RR: 1.4, 95% CI: 0.9-2.3).39 In another study McCredie et al. (1995)40 pooled data from 1,313 cases and 1724 controls from Australia, Denmark, Germany, Sweden and the US, identified between 1989 and 1991. The role of phenacetin-containing and other types of analgesics in the development of renal-cell cancer was studied. Relative risks, adjusted for the effects of age, sex, body-mass index, tobacco smoking and study centre, were not statistically significantly increased with a lifetime consumption of > 0.1 kg phenacetin (or when subjects were subdivided further by amount). According to the authors, these findings do not support the hypothesis that analgesics containing phenacetin increase the risk, although the 20 Phenacetin number of ‘regular’ users and the amount of analgesics consumed were too small to confidently rule out a minor carcinogenic effect of phenacetin. Kreiger et al. (1993) performed a population-based case-control study in Ontario, Canada of risk factors for renal cell carcinoma. Data were collected on 518 case and 1,381 controls identified between 1986 and 1987. In this large study different risk factors for renal cell carcinoma were observed. No association was found between phenacetin-only use (5 cases, 9 controls) and the risk of renal cell carcinoma (OR: 2.5, 95% CI: 0.3-18.5 for males and OR: 1.8, 95% CI: 0.5-7.3 for females) or between acetaminophen-only use and the risk of renal cell carcinoma (OR: 0.8, 95% CI: 0.3-1.7 for males and OR: 0.9, 95% CI: 0.5-2.0 for females), although few subjects used either compound.34 Gago et al. (1999) conducted a population-based case-control study in Los Angeles, US (1,204 cases and equal number controls) to investigate the relationship between sustained use of analgesics and the risk of renal cell carcinoma. Regular use of analgesics (2 or more times a week for 1 months or longer) was a significant risk factor for renal cell carcinoma for all four major classes of analgesics (aspirin, non-steroidal anti-inflammatory agents other than aspirin, acetaminophen and phenacetin). Regular use of phenacetin containing analgesics was associated with an OR of 1.9 (95% CI: 1.3-2.7). A dose-related increase in risk of renal cell carcinoma was observed after further subdivision into different amounts of the maximum weekly dose.32 Bladder cancer Several epidemiological studies 23,25,27,29-31,43 have examined phenacetin and bladder cancer. Two of the case-control studies on renal pelvis and ureter cancer which are already described above, also analysed the risk of phenacetincontaining analgesics consumption for the development of bladder cancer (McCredie et al., 1988; Pommer et al., 1999).37,43 In the population-based case-control study in New South Wales, Australia by McCredie et al. (1988)37(described above), risk for cancer of the bladder was doubled by the consumption of phenacetin (OR: 2.0, 95% CI: 1.1-3.5 for subjects with a lifetime consumption of > 1 kg phenacetin and OR: 2.1, 95% CI: 1.3-3.5 for subjects with a lifetime consumption of > 0.1 kg phenacetin). In the case-control study in Berlin, Germany by Pommer et al. (1999)43 (described above), no association was observed between a lifetime intake of more than 1 kg phenacetin in analgesic mixtures and bladder cancer (OR: 0.75, 95% CI: 0.39-1.43). Carcinogenicity 21 In a population-based case-control study conducted in Los Angeles, California, US by Castelao et al. (2000), 1,514 cases of bladder cancer and an equal number of controls, identified between 1987-1996 were investigated. Regular use of analgesics was not associated with an increased risk of bladder cancer in either man or women. The intake of phenacetin-containing analgesics was positively related to bladder cancer risk in a dose-dependent manner, while the intake of its major metabolite in humans, acetaminophen, was unrelated to risk. Regular use of phenacetin-containing analgesics was not associated with an increased risk of bladder cancer (OR: 1.5, 95% CI: 0.85-2.73).29 In a hospital based case-control study conducted in Spain by Fortuny et al. (2006), the use of non-aspirin non-steroidal anti-inflammatory drugs (NSAID), aspirin, paracetamol (acetaminophen), phenacetin, and metamizol (dipyrone) and risk of bladder cancers was assessed. Data on 958 cases and 1,029 controls, identified between 1997 and 2000 was analysed. A significant reduction in bladder cancer risk was observed for regular users of non-aspirin NSAIDs compared with never users. No evidence of an overall effect for regular use paracetamol or aspirin was observed. Regular use of phenacetin was not associated with an increased risk of bladder cancer (OR: 1.3, 95% CI: 0.3-4.5). However, this estimate was based on only 7 cases and 12 controls.30 In a population-based case-control study conducted in New Hampshire, UK by Fortuny et al. (2007), the influence of phenacetin, other analgesics and NSAID use on the risk of bladder cancer was investigated. Data from 376 cases and 463 controls, identified between 1998 and 2001 was analysed. Elevated OR’s were associated with reported use of phenacetin-containing medications (OR: 2.2, 95% CI: 1.3-3.8 for ever compared to never users), especially with longer duration of use (OR: 3.0, 95% CI: 1.4-6.5 for > 8 years of use).31 3.2 Carcinogenicity studies in animals A group of 30 BD I and BD III rats (age, 100 d) received phenacetin (40-50 mg) daily in the diet (average total, dose 22g). One rat died after a total dose of 10 g and was found to have an osteochondroma. The mean age of death of the treated animals was 770 days, the control animals 750 days. No tumours related to treatment were observed.45 Four groups of 15, 20, 20, and 24 male albino rats were fed with diets containing 0, 0.05, 0.1 or 0.5 % N-hydroxyphenacetin (metabolite of phenacetin) during 73 weeks. Assuming a body weight of 400 grams and a daily food intake of 20 grams, the exposure of N-hydroxyphenacetin was 25, 50, and 250 mg/kg bw/day respectively. Of treated animals 11, 13 and 15 rats were still alive at the 22 Phenacetin time of appearance of the first tumour after 45, 45 and 38 weeks. Of these animals 8/11, 13/13 and 15/15 developed liver tumours (described as hepatocellular carcinomas). None of the control group animals developed tumours. One of the animals fed with 0.1% diet developed a transitional cell carcinoma of the renal pelvis.46 Female SD rats were given 0 or 0.535% phenacetin in the diet for 86 or 110 weeks. Assuming a body weight of 400 grams and a daily food intake of 20 grams the exposure of phenacetin was 268 mg/kg bw/day. In the 86-week study, epithelial hyperplasia of renal papillae was found in 2/24 controls and 21/38 treated animals. In the 110 week study the following changes were observed: Urothelial hyperplasia of the renal papillae in 26 animals, dilatation of vasa recta in 28, and epithelial hyperplasia in 1 animal. In addition, carcinomas of the mammary gland (5/30) and ear duct (4/30; P>0.05) were found in the treated group. In the control group, uroepithelial hyperplasia was found in 5 animals, dilatation of vasa recta in 8 and mammary carcinoma in 1 animal.47 Two groups of SD rats (50 male, 50 female, age 9 wks) were fed a diet containing 1.25 or 2.5% phenacetin for 18 months, followed by a basal diet for 6 months. Assuming a body weight of 400 grams and a daily food intake of 20 grams the exposure of phenacetin was 625 and 1,250 mg/kg bw/day respectively. The control group (65 male and 65 female) were fed with the same basal diet. Among animals surviving for 24 months or dying within 24 months with tumour(s), neoplasms were detected in 27/27 males and 21/27 females fed 2.5%, in 20/22 males and 19/25 females fed 1.25% and in 1/19 males and 6/25 females in the control group. Tumours (benign and malignant) of the nasal cavity were found in 16/27 males and 7/27 females fed 2.5% and in 16/22 males and 6/25 females fed 1.25%. Malignant tumours of the urinary tract were detected in 13/27 males and 4/27 females fed with the high dose and in 1/22 males and 0/25 females fed with the low dose; 2 papillomas were found in females given the high dose. No nasal cavity or urinary tract tumours were seen in controls.48 Two groups of B6C3F1 mice (52 male and female, age 6 weeks) were fed for 96 weeks a diet containing 1.25 or 0.6% phenacetin followed by a basal diet for 8 weeks. Assuming a body weight of 20 grams and a daily food intake of 3 grams the exposure of phenacetin was 1,875 and 900 mg/kg bw/day respectively. The control group of animals (50 mice of each sex) was fed the same basal diet for 104 weeks. All animals were killed at the end of the experiment. The organs were examined histopathologically. Mice that died during the experiment were also autopsied. Phenacetin at a dose of 0.6% induced a significant increased incidence of renal cell adenoma in male mice only. A dose of 1.25% was induced a significant Carcinogenicity 23 increase in both renal cell adenoma and carcinoma in male mice. A clear doseresponse relationship was seen between the doses of phenacetin and the induction of renal cell carcinoma. A statistically significant increased incidence of tumours was found in the liver, lung, skin, hematopoietic system (leukaemia or lymphoma) and occasionally in some other organs.49 Four groups of twenty rats (male Sprague-Dawley, age 6 weeks) were given phenacetin (0, 0.5, 1.0 or 1.5 %) in the diet for 6 or 12 weeks. The 0.5, 1.0 and 1.5 % groups had a real phenacetin intake of 0.78, 1.28 and 1.77 g/kg bw (at week 2 of the experiment) and this intake decreased to 0.31, 0.65 and 1.18 g/kg bw (at week 12).Ten rats of each group were killed at 6 and 12 weeks. One hour before killing a single i.p injection of labelled thymidine was given. To determine to which extent the labelled thymidine was incorporated in the DNA of various tissues, the labelling index was measured. A high labelling index indicates a high cell proliferation. There was a dose-related increase in the labelling index in the urothelium of the bladder and kidney (especially after 6 weeks and 1.0% and 1.5% dose). After 6 weeks the labelling indices were increased in the bladder. After 12 weeks the labelling indices in the bladder were only increased numerically but not statistically significant. In the renal pelvic the labelling index was significantly increased at doses of 1.0 and 1.5 %. At week 12 the majority of rats treated with 1.5% had labelling indices ≥ 2-fold than the control both in kidney and bladder. The increased labelling indices were associated with urothelial hyperplasia (in particular after 6 weeks).50 Twenty male Crl:CDBR rats were treated by gavage with phenacetin during 7 or 14 days. The rats were divided in 4 groups: a control, a low-dose (100 mg/kg bw/day), an intermediate (625 mg/kg bw/day) and a high-dose group (1,250 mg/kg bw/day). One week of phenacetin treatment resulted in dose-related increases in DNA synthesis in both respiratory and olfactory mucosa. The increase observed in the respiratory mucosa was due to inflammatory cells in the lamina propria and not to proliferation of the respiratory epithelial cells. One or two weeks of daily phenacetin treatment resulted in degenerative changes in the olfactory epithelium and necrosis of Bowman’s glands. These changes were associated with increases in cell proliferation in the olfactory epithelium only. Two-week daily gavage treatment of rats with phenacetin at 100, 625 and 1,250 mg/kg/day increased olfactory epithelial cell replication by 62.1, 174 and 763%, respectively.51 Phenacetin was mixed in the feed at a concentration of 0.7 or 1.4% and administered to transgenic CB6F1-rasH2 mice and non-transgenic, wildtype (non-Tg, WT) mice during 24 weeks. Assuming a body weight of 20 grams and a daily food intake of 3 grams the exposure to phenacetin was 1,050 and 2,100 24 Phenacetin mg/kg bw/day respectively. Phenacetin induced spleen haemangiosarcoma and lung adenomas in the rasH2mice but not in the non-Tg mice. Lung adenomas (12 in exposed versus 2 in control) and spleen hemangiosarcomas (6/0) were found in male rasH2 treated with 1.4% phenacetin in the feed. This incidence was significant higher than in the corresponding non-Tg mice.52 P53+/- transgenic mice were given phenacetin by daily gavage with dose of 100, 200 and 350 mg/kg bw/day suspended in 0.5% methylcellulose during 26 weeks. In a separate study the mice were given a dose of 0.14, 0.7 and 1.4% phenacetin in the diet. Control and high-dose groups of wild-type mice were included in both studies. No increase in treatment-related tumour incidence was found after 26 week of treatment.53 The transgenic Tg.AC mice strain is able to respond to dermal application with development of squamous-cell papillomas of the skin. Phenacetin was administered topically (0, 0.08, 0.4 and 2 mg, daily) and in the diet (0, 12, 60, 300 ppm) during 26 weeks. Phenacetin was negative by both routes of exposure.54 Phenacetin was administered in the feed (0, 0.1, 0.25, 0.5, or 0.75% w/w) to transgenic Xpa-/- mice (15 male, 15 female), to double transgenic Xpa-/-/p53+/mice (15 male, 15 female) and to wild type (WT) C57BL/6 mice (15 male, 15 female). Assuming a body weight of 20 grams and a daily food intake of 3 grams the exposure of phenacetin was 150, 375, 750, 1,125 mg/kg bw/day respectively. The exposure to phenacetin was 39 weeks for all groups. At the end of the experiment renal proximal tubular hyperplasia was observed in two high-dose Xpa-/- males and in one Xpa-/-/p53+/- male mouse. A tubular adenoma was found in a Xpa-/-/p53+/- female mouse. In all male and female transgenic, but not the WT mice, multifocal karyomegaly in the proximal renal tubules was found. In addition, olfactory epithelial degeneration was observed in the nose of most male and female transgenic and WT mice of the high-dose groups.55 Phenacetin had the ability to induce morphological transformation in cultured C3H/10T1/2 clone 8 mouse embryo cells (10T1/2 cells). Treatment of the 10T1/2 cells with 0.5, 1.0, and 2.0 mg/ml phenacetin caused a dose-dependent decrease in plating efficiency and a dose-dependent increase in type II morphologically transformed foci.56 Phenacetin tested in the Syrian hamster embryo transformation assay gave negative results. The highest concentration phenacetin tested was 500 µg/ml phenacetin. Phenacetin above a concentration level of 500 µg/ml was insoluble in the medium with DMSO.57 Carcinogenicity 25 In an initiation-promotion experiment male F344 rats (6 weeks of age) were divided in two groups of 20 and one of 10 rats. The two groups of 20 rats were pretreated with 0.1% DHPN in drinking water and 3.0% uracil in the diet during 4 weeks. DHPN (dihydroxy-di-N-propylnitrosamine) is a carcinogen which is known to induce tumours of the renal pelvis, renal tubular cells and urinary bladder in rats. One week after cessation, one group received basal diet and one group received a diet containing 2.0% phenacetin (average intake 1,145 mg/kg/ day) during the following 35 weeks. The group of 10 animals was given, during the same period, a diet with 2.0% phenacetin (average intake 1,068 mg/kg/day) without the initial combination treatment of DHPN and uracil. The occurrence of renal cell tumours was increased in the group given phenacetin (9/20) as compared with the DHPN + uracil alone control (1/19). In the urinary bladder, phenacetin treatment was associated with increased incidence of preneoplastic or neoplastic lesions. The group of animals, treated with phenacetin alone, without the pretreatment, induced simple hyperplasias of the urinary bladder at high incidence.58 26 Phenacetin Chapter 4.1 4 Mode of action Genotoxic mode of action More details of these studies have been summarized in Annex H. 4.1.1 Gene mutation assays In vitro Phenacetin was not mutagenic in several bacterial models in the presence or absence of rat or mouse liver microsome preparations: the models included a repair test in Bacillus subtillus59 and reverse mutation test in Salmonella typhimurium TA1535, TA 1537, TA98 and TA 10060,61, Escherichia coli K 12/343/1361, and B. subtilis TKJ 5211.59 Positive bacterial mutagenic results have been obtained in S. typhimurium TA 100 in the presence of hamster, but not rat, liver post-mitochondrial supernatant of Aroclor-treated animals.62-64 Phenacetin led to an increase in the mutant frequency in Salmonella typhimurium TA 100 in the presence of a hamster liver metabolic activation.65,66 In the hprt test phenacetin induced an increase in the mutant frequency in V79 Chinese hamster cells in vitro in the presence of hamster liver microsome preparations.65,67. Mode of action 27 In vivo Phenacetin was negative in an intrasanguineous host-mediated assay with E.coli K 12 in NMRI mice given 2 mmol/kg intraperitoneally. Phenacetin did not induce an increased frequency of sex-linked recessive lethals in Drosophila melanogaster. Phenacetin was given in the feed of DNA repair deficient (Xpa-/- and Xpa-/-/ Trp53+/-) mice and wild type (WT) carrying the IacZ (0.75% w/w, during 0, 4, 8, or 12 weeks). Xpa-/- mice lack the normal nucleotide excision repair pathway. Due to this deficiency, these mice are more sensitive to genotoxic compounds than wild type mice. Phenacetin exposure induced an increase in the lacZ mutant frequency in the kidney of WT, Xpa-/- and Xpa-/-/Trp53+/- mice as compared with concurrent untreated controls of the wild type C57BL/6 mice. The increase in Xpa-/- and Xpa-/-/Trp53+/- mice was stronger than in WT mice. A minor and negative response was found in the liver and the spleen, respectively. The observed phenacetin-induced mutant frequency was higher in male than in female mice.68 4.1.2 Cytogenetic assays In vitro Phenacetin induced DNA fragmentations in an acellulair test-system with λ DNA but not with calf thymus DNA.69 In vivo No data were available on the genetic and related effects of phenacetin in humans. The results of studies on the induction of chromosomal aberrations, sister chromatid exchanges and micronuclei in rodents treated with phenacetin in vivo were equivocal.61,70 Phenacetin exposure did not result in an enhanced number of micronucleated erythrocytes in the bone marrow of NMRI mice given 2 x 5 mmol/kg bw intraperitoneally.61 Following in vivo treatment, the alkaline elution assay showed no increase of DNA damage in bone-marrow cells of i.p-treated mice or in liver cells of rats treated by gavage. However, an increase of DNA damage was observed in liver of rats after i.p. administration of phenacetin and in kidney of rats receiving 28 Phenacetin phenacetin by gavage.65 Sister chromatid exchanges were seen in mice (i.p, 330 mg/kg bw) treated with phenacetin. This increase of SCE was weak but statistically significant.65 The micronucleus bone marrow test showed a positive response in mice given phenacetin i.p. Phenacetin doses of 37.5, 75, 150, 300, 400 and 600 mg/kg bw/day were administered only once or multiple times (2-4) to CD-1 mice. Positive responses were seen at 600 mg/kg/day after single and triple dosing and at 400 and 600 mg/kg/day after double dosing.71,72 A single dose of phenacetin of 0, 2, 5, 50 and 100 mg/kg given i.p to SJL Swiss mice resulted in a moderate but significant increase of cells with micronuclei compared with the control group.73 The micronucleus assay with peripheral reticulocytes from phenacetin-treated mice (CD-1 and MS/Ae strain) was negative after a single dose of 400, 600 and 800 mg/kg bw(24 h after i.p). Positive results were obtained with 600 and 800 mg/kg bw after 48 h. Double treatment (24 h between treatments) enhanced the responses. A dose response was obtained for all different sample times. In this same experiment CD-1 mice treated with phenacetin (i.p, 600 mg/kg bw, single and double treatment) gave a positive result in the micronucleus test in bone marrow cells.74 Phenacetin was administered to rats (Sprague-Dawley) with doses of 500, 1,000 and 2,000 mg/kg bw/day during 2 days or 250, 500, 750, 1,000 mg/kg bw/day during 14 days. Blood samples were taken on day 1, 3, 6, 9, 12 and 15 for the micronucleus assay with peripheral reticulocytes. In the 14-day test, phenacetin increased the frequency of micronucleated reticulocytes in peripheral blood at 500 mg/kg bw/day starting from day 9, and at 750 and 1,500 mg/kg bw/day starting from day 6. In the test with 2 days application the frequencies of micronucleated reticulocytes increased at 1,000 and 2,000 mg/kg bw/day. In the test with 14 days application the micronucleus assay in the bone marrow showed a positive dose-related response.75 4.1.3 Miscellaneous In vitro Hepatocytes isolated from mouse, hamster, rat and guinea pig showed no marked increase in unscheduled DNA synthesis (UDS) after exposure to phenacetin.76 After treatment with phenacetin, mouse L-cells gave positive results using a DNA-synthesis inhibition test system.77 An increase in DNA damage measured by the alkaline elution assay was not observed when human and rat hepatocytes were treated with phenacetin in vitro.78 Mode of action 29 30 Phenacetin Chapter 5.1 5 Classification Evaluation of data on carcinogenicity and genotoxicity The Committee is aware that in most of the epidemiological studies described above the effect of phenacetin may be influenced by other analgetic comedications, by selection bias, especially in the hospital-based case-control studies, and recall bias. However, the Committee is also of the opinion that the epidemiological evidence cannot exclude that phenacetin-containing analgetics are part of the etiology of renal pelvic, urothelial and bladder cancer. However, the evidence is considered sufficient by the Committee. For bladder cancer the evidence does not support such a relationship. Based on the available information the Committee concludes that there is sufficient evidence for carcinogenicity of phenacetin to humans. Phenacetin induced tumours of the urinary tract (in mice and rats) and nasal cavity (in rat) when given orally. New published data consisted of 9 not standard carcinogenicity studies, which support this conclusion. Three of these studies with rats gave insight in the mechanism of the damage induced by phenacetin. They gave evidence of DNA damage in the bladder or nasal mucosa. Four other studies used transgenic mice. In two of these studies, the transgenic mice showed increased lung, spleen and kidney tumours compared to wild type mouse. The two other studies are transformation tests with mouse-embryo and hamster embryo cells, of which only the study in mouse-embryo showed increased transformation. Considering the available animal data, the Committee concludes Classification 31 that there is sufficient evidence for carcinogenicity of phenacetin to animals. In addition, the Committee is aware that both animal data and the human data show a relationship beween phenacetin and cancer of the kidney. This relationship was even more supported by the observation that phenacetin increased the lacZ mutant frequency in kidney of transgenic mice. Such an analogy in cancer development in man and animal on the level of a specific organ supports the role of phenacetin as a carcinogen. Phenacetin was negative in almost all in vitro bacterial mutagenicity tests. On the other hand, DNA damage was observed in mammalian cells in vitro and in vivo. Phenacetin induced inhibition of DNA synthesis and an increase in the mutant frequency in a gene mutation assay with mammalian cells when hamster but not rat S9 mix was used as metabolic activation. The positive findings in vitro were confirmed in in vivo genotoxicity tests. Phenacetin was positive in several micronucleus tests as well as in a gene mutation test with transgenic animals; in several studies a clear dose-response relationship was observed. Therefore, it can be concluded that phenacetin is a stochastic genotoxic compound. 5.2 Recommendation for classification The Committee concludes that phenacetin is carcinogenic to humans and recommends classifying the substance in category 1A.* Moreover, the Committee concludes that phenacetin has a stochastic genotoxic working mechanism. * According to the classification system of the Health Council (see Annex I). 32 Phenacetin References 1 IARC. Overall evaluations of carcinogenicity: an updating of IARC Monographs volumes 1 to 42. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 1987; Suppl 7: 1-440. 2 IARC. Some miscellaneous pharmaceutical substances. IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans: 1977; 13: 1-255. 3 IARC. Some miscellaneous pharmaceutical substances. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 1980; 24: 135-161. 4 IARC. A review of human carcinogens: Pharmaceuticals. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 2011; 100A: 370-400. 5 ESIS. European Chemical Substances Information System (http://esis.jrc.europa.eu/), accessed September. 2012. 6 HSDB. Hazardeous Substances Data Bank (http://toxnet.nlm.nih.gov/), accessed September. 2012. 7 Blohme I, Johansson S. Renal pelvic neoplasms and atypical urothelium in patients with end-stage analgesic nephropathy. Kidney Int 1981; 20(5): 671-5. 8 Christensen TE, Ladefoged J. [Uroepithelial tumors in patients with contracted kidneys and massive abuse of analgesics (phenacetin)]. Ugeskr Laeger 1979; 141(51): 3522-3524. 9 Gonwa TA, Corbett WT, Schey HM, Buckalew VM, Jr. Analgesic-associated nephropathy and 10 Kliment J. [Renal pelvis tumours and abuse of analgesics (author's transl)]. Bratisl Lek Listy 1979; 11 Marek J, Hradec E. Chronic sclerosing ureteritis and nephrogenic adenoma of the ureter in analgesic transitional cell carcinoma of the urinary tract. Ann Intern Med 1980; 93(2): 249-252. 72(6): 708-713. abuse. Pathol Res Pract 1985; 180(5): 569-575. References 33 12 Mihatsch M J, Manz T, Knusli C, Hofer H O, Rist M, Guetg R et al. Phanacetin abuse III. Malignant urinary tract tumors in phenacetin abuse in Basle 1963-1977 Original Title: Phenacetinabusus III. Maligne Harnwegtumoren bei Phenacetinabusus in Basel 1963-1977. Schweiz Med Wochenschr 1980; 110(7): 255-264. 13 Mihatsch MJ, Brunner FP, Korteweg E, Rist M, Dalquen P, Thiel G. [Phenacetin abuse. VII: Urinary tract tumors in dialysis patients and patients with kidney grafts]. Schweiz Med Wochenschr 1982; 112(42): 1468-1472. 14 Orell SR, Nanra RS, Ferguson NW. Renal pelvic carcinoma in the Hunter Valley. Med J Aust 1979; 2(10): 524, 555-524, 557. 15 Porpaczy P, Schramek P. Analgesic nephropathy and phenacetin-induced transitional cell carcinoma analysis of 300 patients with long-term consumption of phenacetin-containing drugs. Eur Urol 1981; 7(6): 349-354. 16 Syre G, Matejka M. [Abuse of phenacetin-containing analgesics and carcinoma of the renal pelvis (author's transl)]. Wien Klin Wochenschr 1981; 93(13): 420-423. 17 Anderstrom C, Johansson SL, Pettersson S, Wahlqvist L. Carcinoma of the ureter: a clinicopathologic study of 49 cases. J Urol 1989; 142(2 Pt 1): 280-3. 18 Burnett KR, Miller JB, Greenbaum EI. Transitional cell carcinoma: rapid development in phenacetin abuse. AJR Am J Roentgenol 1980; 134(6): 1259-61. 19 Holmäng SaJSL. Synchronous bilateral ureteral and renal pelvic carcinomas. Cancer 2004; 101(4): 741-747. 20 Lornoy W, Morelle V, Becaus I, Fonteyne E, Mestdagh J, Thienpont L et al. Malignant uroepithelial tumors of the upper urinary tract in sixteen patients with analgesic nephropathy. Acta Clin Belg 1980; 35(3): 140-7. 21 Petersen I, Ohgaki H, Ludeke BI, Kleihues P. p53 Mutationen in Phenazetin-induzierten Urothelkarzinomen. [p53 mutation in phenacetin-induced urothelial carcinomas]. Verh Dtsch Ges Pathol 1993; 77: 252-5. 22 Steffens J, Nagel R. Tumours of the renal pelvis and ureter. Observations in 170 patients. Br J Urol 1988; 61(4): 277-83. 23 McCredie M, Ford JM, Taylor JS, Stewart JH. Analgesics and cancer of the renal pelvis in New South Wales. Cancer 1982; 49(12): 2617-2625. 24 McCredie M, Stewart JH, Ford JM. Analgesics and tobacco as risk factors for cancer of the ureter and renal pelvis. J Urol 1983; 130(1): 28-30. 25 McCredie M, Stewart JH, Ford JM, MacLennan RA. Phenacetin-containing analgesics and cancer of the bladder or renal pelvis in women. Br J Urol 1983; 55(2): 220-224. 26 McLaughlin JK, Mandel JS, Blot WJ, Schuman LM, Mehl ES, Fraumeni JF, Jr. A population--based case--control study of renal cell carcinoma. J Natl Cancer Inst 1984; 72(2): 275-284. 27 Piper JM, Tonascia J, Matanoski GM. Heavy phenacetin use and bladder cancer in women aged 20 to 49 years. N Engl J Med 1985; 313(5): 292-295. 34 Phenacetin 28 Piper JM, Matanoski GM, Tonascia J. Bladder cancer in young women. Am J Epidemiol 1986; 123(6): 1033-1042. 29 Castelao JE, Yuan JM, Gago DM, Yu MC, Ross RK. Non-steroidal anti-inflammatory drugs and bladder cancer prevention. Br J Cancer 2000; 82(7): 1364-1369. 30 Fortuny J, Kogevinas M, Garcia-Closas M, Real FX, Tardon A, Garcia-Closas R et al. Use of analgesics and nonsteroidal anti-inflammatory drugs, genetic predisposition, and bladder cancer risk in Spain. Cancer Epidemiol Biomarkers Prev 2006; 15(9): 1696-1702. 31 Fortuny J, Kogevinas M, Zens MS, Schned A, Andrew AS, Heaney J et al. Analgesic and antiinflammatory drug use and risk of bladder cancer: a population based case control study. BMC Urol 2007; 7: 13. 32 Gago Dominguez M, Yuan JM, Castelao JE, Ross RK, Yu MC. Regular use of analgesics is a risk factor for renal cell carcinoma. Br J Cancer 1999; 81(3): 542-8. 33 Jensen OM, Knudsen JB, Tomasson H, Sorensen BL. The Copenhagen case-control study of renal pelvis and ureter cancer: role of analgesics. Int J Cancer 1989; 44(6): 965-8. 34 Kreiger N, Marrett LD, Dodds L, Hilditch S, Darlington GA. Risk factors for renal cell carcinoma: results of a population-based case-control study. Cancer Causes Control 1993; 4(2): 101-110. 35 Linet MS, Chow WH, McLaughlin JK, Wacholder S, Yu MC, Schoenberg JB et al. Analgesics and cancers of the renal pelvis and ureter. Int J Cancer 1995; 62(1): 15-8. 36 McCredie M, Stewart JH, Carter JJ, Turner J, Mahony JF. Phenacetin and papillary necrosis: independent risk factors for renal pelvic cancer. Kidney Int 1986; 30(1): 81-4. 37 McCredie M, Stewart JH. Does paracetamol cause urothelial cancer or renal papillary necrosis? Nephron 1988; 49(4): 296-300. 38 McCredie M, Coates MS, Ford JM, Disney AP, Auld JJ, Stewart JH. Geographical distribution of cancers of the kidney and urinary tract and analgesic nephropathy in Australia and New Zealand. Aust N Z J Med 1990; 20(5): 684-8. 39 McCredie M, Stewart JH, Day NE. Different roles for phenacetin and paracetamol in cancer of the kidney and renal pelvis. Int J Cancer 1993; 53(2): 245-9. 40 McCredie M, Pommer W, McLaughlin JK, Stewart JH, Lindblad P, Mandel JS et al. International renal-cell cancer study. II. Analgesics. Int J Cancer 1995; 60(3): 345-9. 41 McLaughlin JK, Blot WJ, Mehl ES, Fraumeni JF. Relation of analgesic use to renal cancer: population-based findings. Natl Cancer Inst Monogr 1985; 69: 217-22. 42 McLaughlin JK, Gao YT, Gao RN, Zheng W, Ji BT, Blot WJ et al. Risk factors for renal-cell cancer in Shanghai, China. Int J Cancer 1992; 52(4): 562-5. 43 Pommer W, Bronder E, Klimpel A, Helmert U, Greiser E, Molzahn M. Urothelial cancer at different tumour sites: role of smoking and habitual intake of analgesics and laxatives. Results of the Berlin Urothelial Cancer Study. Nephrol Dial Transplant 1999; 14(12): 2892-7. 44 Stewart JH, Hobbs JB, McCredie MR. Morphologic evidence that analgesic-induced kidney pathology contributes to the progression of tumors of the renal pelvis. Cancer 1999; 86(8): 1576-1582. References 35 45 Schmal D, REITER A. [Absence of carcinogenic effect in phenacetin.]. Arzneimittelforschung 1954; 4(6): 404-405. 46 Calder IC, Goss DE, Williams PJ, Funder CC, Green CR, Ham KN et al. Neoplasia in the rat induced by N-hydroxyphenacetin, a metabolite of phenacetin. Pathology 1976; 8(1): 1-6. 47 Johansson S, Angervall L. Urothelial changes of the renal papillae in Sprague-Dawley rats induced by long term feeding of phenacetin. Acta Pathol Microbiol Scand [A] 1976; 84(5): 375-383. 48 Isaka H, Yoshii H, Otsuji A, Koike M, Nagai Y, Koura M et al. Tumors of Sprague-Dawley rats induced by long-term feeding of phenacetin. Gann 1979; 70(1): 29-36. 49 Nakanishi K, Kurata Y, Oshima M, Fukushima S, Ito N. Carcinogenicity of phenacetin: long-term feeding study in B6c3f1 mice. Int J Cancer 1982; 29(4): 439-44. 50 Johansson SL, Radio SJ, Saidi J, Sakata T. The effects of acetaminophen, antipyrine and phenacetin on rat urothelial cell proliferation. Carcinogenesis 1989; 10(1): 105-11. 51 Bogdanffy MS, Mazaika TJ, Fasano WJ. Early cell proliferative and cytotoxic effects of phenacetin on rat nasal mucosa. Toxicol Appl Pharmacol 1989; 98(1): 100-12. 52 Usui T, Mutai M, Hisada S, Takoaka M, Soper KA, McCullough B et al. CB6F1-rasH2 mouse: overview of available data. Toxicol Pathol 2001; 29 Suppl: 90-108. 53 Storer RD, French JE, Haseman J, Hajian G, LeGrand EK, Long GG et al. P53+/- hemizygous knockout mouse: overview of available data. Toxicol Pathol 2001; 29 Suppl: 30-50. 54 Eastin WC, Mennear JH, Tennant RW, Stoll RE, Branstetter DG, Bucher JR et al. Tg.AC genetically altered mouse: assay working group overview of available data. Toxicol Pathol 2001; 29 Suppl: 6080. 55 Lina BA, Woutersen RA, Bruijntjes JP, van Benthem J, van den Berg JA, Monbaliu J et al. Evaluation of the Xpa-deficient transgenic mouse model for short-term carcinogenicity testing: 9-month studies with haloperidol, reserpine, phenacetin, and D-mannitol. Toxicol Pathol 2004; 32(2): 192-201. 56 Patierno SR, Lehman NL, Henderson BE, Landolph JR. Study of the ability of phenacetin, acetaminophen, and aspirin to induce cytotoxicity, mutation, and morphological transformation in C3H/10T1/2 clone 8 mouse embryo cells. Cancer Res 1989; 49(4): 1038-44. 57 Mauthe RJ, Gibson DP, Bunch RT, Custer L. The syrian hamster embryo (SHE) cell transformation assay: review of the methods and results. Toxicol Pathol 2001; 29 Suppl: 138-46. 58 Shibata MA, Sano M, Hagiwara A, Hasegawa R, Shirai T. Modification by analgesics of lesion development in the urinary tract and various other organs of rats pretreated with dihydroxy-di-Npropylnitrosamine and uracil. Jpn J Cancer Res 1995; 86(2): 160-7. 59 Tanooka H. Development and applications of Bacillus subtilis test systems for mutagens, involving DNA-repair deficiency and suppressible auxotrophic mutations. Mutat Res 1977; 42(1): 19-31. 60 Shudo K, Ohta T, Orihara Y, Okamoto T, Nagao M, Takahashi Y et al. Mutagenicities of phenacetin and its metabolites. Mutat Res 1978; 58(2-3): 367-370. 61 King MT, Beikirch H, Eckhardt K, Gocke E, Wild D. Mutagenicity studies with x-ray-contrast media, analgesics, antipyretics, antirheumatics and some other pharmaceutical drugs in bacterial, Drosophila and mammalian test systems. Mutat Res 1979; 66(1): 33-43. 36 Phenacetin 62 Camus AM, Friesen M, Croisy A, Bartsch H. Species-specific activation of phenacetin into bacterial mutagens by hamster liver enzymes and identification of N-hydroxyphenacetin O-glucuronide as a promutagen in the urine. Cancer Res 1982; 42(8): 3201-8. 63 Dunkel VC, Simmon VF. Mutagenic activity of chemicals previously tested for carcinogenicity in the National Cancer Institute bioassay program. IARC Sci Publ 1980;(27): 283-301. 64 Weinstein D, Katz M, Kazmer S. Use of rat/hamster S-9 mixture in the Ames mutagenicity assay. Environ Mutagen 1981; 3(1): 1-9. 65 De Flora S, Russo P, Pala M, Fassina G, Zunino A, Bennicelli C et al. Assay of phenacetin genotoxicity using in vitro and in vivo test systems. J Toxicol Environ Health 1985; 16(3-4): 355-77. 66 Oldham JW, Preston RF, Paulson JD. Mutagenicity testing of selected analgesics in Ames Salmonella strains. J Appl Toxicol 1986; 6(4): 237-43. 67 Fassina G, ABBONDANDOLO A, MARIANI L, TANINGHER M, Parodi S. Mutagenicity in V79 cells does not correlate with carcinogenicity in small rodents for 12 aromatic amines. J Tox Env Health 1990; 29(1): 109-130. 68 Luijten M, Speksnijder EN, van Alphen N, Westerman A, Heisterkamp SH, van Benthem J et al. Phenacetin acts as a weak genotoxic compound preferentially in the kidney of DNA repair deficient Xpa mice. Mutat Res 2006; 596(1-2): 143-150. 69 Adams SP, Laws GM, Storer RD, DeLuca JG, Nichols WW. Detection of DNA damage induced by human carcinogens in acellular assays: Potential application for determining genotoxic mechanisms. Mutat Res ; 1996; 368(3-4): 235-248. 70 Granberg Ohman I, Johansson S, Hjerpe A. Sister-chromatid exchanges and chromosomal aberrations in rats treated with phenacetin, phenazone and caffeine. Mutat Res 1980; 79(1): 13-8. 71 Sutou S, Kondo M, Mitsui Y. Effects of multiple dosing of phenacetin in the micronucleus test. Mutat Res 1990; 234(3-4): 183-6. 72 Sutou S, Mitui Y, Toda S, Sekijima M, Kawasaki K, Ando N et al. Effect of multiple dosing of phenacetin on micronucleus induction: a supplement to the international and Japanese cooperative studies. Mutat Res 1990; 245(1): 11-4. 73 Sicardi SM, Martiarena JL, Iglesias MT. Mutagenic and analgesic activities of aniline derivatives. J Pharm Sci ; 1991; 80(8): 761-764. 74 Higashikuni N, Baba T, Nakamura T, Sutou S. The micronucleus test with peripheral reticulocytes from phenacetin-treated mice. Mutat Res 1992; 278(2-3): 159-64. 75 Asanami S, Shimono K, Sawamoto O, Kurisu K, Uejima M. The suitability of rat peripheral blood in subchronic studies for the micronucleus assay. Mutat Res 1995; 347(2): 73-8. 76 Holme JA, Soderlund E. Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes. Mutat Res 1986; 164(3): 167-75. 77 Gotoh S, Higashi K, Miyata Y, Nishi C, Sakamoto Y. Screening for carcinogens by DNA-synthesis inhibition test using mouse L-cells. J UOEH 1983; 5(2): 147-53. References 37 78 Robbiano L, Allavena A, Bagarolo C, Martelli A, Brambilla G. Comparison in human and rat hepatocytes of the DNA-damaging activity of five chemicals probably carcinogenic to humans. Toxicol in Vitro; 1994; 8(1): 131-137. 79 Guideline to the classification of carcinogenic compounds. Health Council of The Netherlands, editor. The Hague, The Netherlands: 2010: publication no. A10/07E. 38 Phenacetin A Request for advice B The Committee C The submission letter D Comments on the public review draft E IARC Monograph F Human data G Animal data H Genotoxicity data I Carcinogenic classification of substances by the Committee Annexes 39 40 Phenacetin Annex A Request for advice In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs and Employment wrote: Some time ago a policy proposal has been formulated, as part of the simplification of the governmental advisory structure, to improve the integration of the development of recommendations for health based occupation standards and the development of comparable standards for the general population. A consequence of this policy proposal is the initiative to transfer the activities of the Dutch Expert Committee on Occupational Standards (DECOS) to the Health Council. DECOS has been established by ministerial decree of 2 June 1976. Its primary task is to recommend health based occupational exposure limits as the first step in the process of establishing Maximal Accepted Concentrations (MAC-values) for substances at the work place. In an addendum, the Minister detailed his request to the Health Council as follows: The Health Council should advice the Minister of Social Affairs and Employment on the hygienic aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air quality at the work place. This implies: • A scientific evaluation of all relevant data on the health effects of exposure to substances using a criteria-document that will be made available to the Health Council as part of a specific request Request for advice 41 for advice. If possible this evaluation should lead to a health based recommended exposure limit, or, in the case of genotoxic carcinogens, a ‘exposure versus tumour incidence range’ and a calculated concentration in air corresponding with reference tumour incidences of 10-4 and 10-6 per year. • The evaluation of documents review the basis of occupational exposure limits that have been recently established in other countries. • Recommending classifications for substances as part of the occupational hygiene policy of the government. In any case this regards the list of carcinogenic substances, for which the classification criteria of the Directive of the European Communities of 27 June 1967 (67/548/ EEG) are used. • Reporting on other subjects that will be specified at a later date. In his letter of 14 December 1993, ref U 6102/WP/MK/459, to the Minister of Social Affairs and Employment the President of the Health Council agreed to establish DECOS as a Committee of the Health Council. The membership of the Committee is given in Annex B. 42 Phenacetin Annex B The Committee • • • • • • • • R.A. Woutersen, chairman Toxicologic Pathologist, TNO Innovation for Life, Zeist; Professor of Translational Toxicology, Wageningen University and Research Centre, Wageningen J. van Benthem Genetic Toxicologist, National Institute for Public Health and the Environment, Bilthoven P.J. Boogaard Toxicologist, SHELL International BV, The Hague G.J. Mulder Emeritus Professor of Toxicology, Leiden University, Leiden Ms M.J.M. Nivard Molecular Biologist and Genetic Toxicologist, Leiden University Medical Center, Leiden G.M.H. Swaen Epidemiologist, Dow Chemicals NV, Terneuzen E.J.J. van Zoelen Professor of Cell Biology, Radboud University Nijmegen, Nijmegen G.B. van der Voet, scientific secretary Health Council of the Netherlands, The Hague The Committee 43 The Health Council and interests Members of Health Council Committees are appointed in a personal capacity because of their special expertise in the matters to be addressed. Nonetheless, it is precisely because of this expertise that they may also have interests. This in itself does not necessarily present an obstacle for membership of a Health Council Committee. Transparency regarding possible conflicts of interest is nonetheless important, both for the chairperson and members of a Committee and for the President of the Health Council. On being invited to join a Committee, members are asked to submit a form detailing the functions they hold and any other material and immaterial interests which could be relevant for the Committee’s work. It is the responsibility of the President of the Health Council to assess whether the interests indicated constitute grounds for nonappointment. An advisorship will then sometimes make it possible to exploit the expertise of the specialist involved. During the inaugural meeting the declarations issued are discussed, so that all members of the Committee are aware of each other’s possible interests. 44 Phenacetin Annex C The submission letter Subject Our reference Your Reference Enclosed Date : Submission of the advisory report Phenacetin : U-7412/BvdV/fs/246-C17 : DGV/MBO/U-932342 :1 : November 13, 2012 Dear State Secretary, I hereby submit the advisory report on the effects of occupational exposure to Phenacetin. This advisory report is part of an extensive series in which carcinogenic substances are classified in accordance with European Union guidelines. This involves substances to which people can be exposed while pursuing their occupation. The advisory report was prepared by the Subcommittee on the Classification of Carcinogenic Substances, a permanent subcommittee of the Health Council’s Dutch Expert Committee on Occupational Safety (DECOS). The advisory report has been assessed by the Health Council’s Standing Committee on Health and the Environment. The submission letter 45 I have today sent copies of this advisory report to the State Secretary of Infrastructure and the Environment and to the Minister of Health, Welfare and Sport, for their consideration. Yours sincerely, (signed) Professor W.A. van Gool President 46 Phenacetin Annex D Comments on the public review draft A draft of the present report was released in June 2012 for public review. No comments were received on the draft document. Comments on the public review draft 47 48 Phenacetin Annex E IARC Monograph Volume 100A, 2011 (excerpt from Phenacetin, pp397-400) Phenacetin was considered by previous IARC Working Groups in 1976 and 1980. Analgesic mixtures containing phenacetin were considered by a previous IARC Working Group in 1987. Since that time, new data have become available, these have been incorporated in the Monograph, and taken into consideration in the present evaluation. 5 Evaluation There is sufficient evidence in humans for the carcinogenicity of analgesic mixtures containing phenacetin. Analgesic mixtures containing phenacetin cause cancer of the renal pelvis, and of the ureter. There is limited evidence in experimental animals for the carcinogenicity of analgesic mixtures containing phenacetin. There is sufficient evidence in humans for the carcinogenicity of phenacetin. Phenacetin causes cancer of the renal pelvis, and of the ureter. There is sufficient evidence in experimental animals for the carcinogenicity of phenacetin. Analgesic mixtures containing phenacetin are carcinogenic to humans (Group 1). Phenacetin is carcinogenic to humans (Group 1). IARC Monograph 49 For the overall evaluation of phenacetin, the Working Group took into consideration that tumours of the renal pelvis and ureter are not known to result from the other components of the analgesic mixtures used in most countries; namely, aspirin, codeine phosphate, and caffeine. 50 Phenacetin Annex F Human data Human case-control studies of phenacetin exposure and different forms of cancer (published after the IARC publication of 1987). reference design/population results confounding remarks factors risk ratio(95% CI) exposure cases / phenacetin controla containing drugs renal pelvic cancer McLaugh population-based case- never m 24/232 OR 1 adjusted for age the separate effects lin et al., control, Minneapolis, f 12/147 OR 1 and cigarette of the analgesics 198541 ever m 26/196 OR 1.2 (0.6-2.4) smoking. could not be US adequately f 9/100 OR 1.3 (0.5-3.4) OR 1.1 (0.6-2.3) assessed because (74 cases and 697 irregular m 21/175 f 12/122 OR 1.1 (0.4-3.2) most long-term controls, identified regular ≤36 mo m 1/17 OR 0.5 (0.02-3.9) users took both between 1974-1979) phenacetin and f 1/12 OR 1.8 (0.4-22.0) regular>36 mo m 4/4 OR 8.1 (1.2-62.2) acetaminophenf 2/10 OR 4.2 (0.4-42.0) containing products McCredie hospital-based case32/672 adjusted for sex no consumption et al., control, Sidney, New (lifetime exposure 198636 South Wales, Australia < 1kg) (66 cases and 751 lifetime exposure > 27/35 RR 20 (12-34) controls, identified 1 kg with RPN between 1970-1982) lifetime exposure > 7/44 RR 3.6 (1.6-8.1) 1 kg absence of RPN Human data 51 ≥ 1 kg / lifetime 33/54 OR 7.9 (4.6-13.8) > 0.1 kg / lifetime 40/636 OR 5.7 (3.2-10.0) non-consumers < 2.04 kg/ lifetime 2.04-6.87 kg/ lifetime > 6.88 kg/ lifetime consumption of aspirin or phenacetin Stewart et “blinded” < 1 kg / lifetime al., histopathological 1.0-4.9 kg / 199944 review of cases from lifetime population- based case- 5.0-9.9 kg / control study, New lifetime South Wales, Australia ≥ 10.0 kg / lifetime 76/474 12/16 16/16 OR 1 OR 5.2 (2.2-12.4) OR 8.3 (3.4-20.5) 42/17 OR 18.5 (8.7-39.9) 20/37 6/5 RR 1.0 RR 1.9 (0.5-7.3) 5/4 RR 2.1 (0.5-8.9) 17/5 RR 5.6 (1.8-18) Pommer et al., 199943 no/rare analgesic intake > 1.0 kg / lifetime 20/19 OR 1.0 7/2 OR 5.3 (0.3-81) adjusted never used ever used 31/113 9/55 13/12 17/15 31/113 9/55 13/12 17/15 6/7 2/3 5/2 7/7 4/4 6/4 385/369 21/23 9/12 RR 1.0 RR 1.0 RR 2.4 (0.9-6.8) RR 4.2 (1.5-12.3) RR 1.0 RR 1.0 RR 3.9 (1.7-9.1) RR 6.9 (2.7-17.7) RR 3.1 (1.0-9.6) RR 6.1 (1.5-25.6) RR 9.1 (2.2-38) RR 6.1 (1.9-20.0) RR 2.4 (0.4-14.5) RR 9.2 (2.5-33) OR 1.0 OR 0.8 (0.4-1.6) OR 0.3 (0.3-2.1) McCredie population-based caseet al., control, New South 198837 Wales, Australia (73 cases and 688 controls, identified between 1980-1982) McCredie population-based caseet al., control, New South 199339 Wales, Australia (147 cases and 523 controls identified in 1989-1990) hospital-based and population-based case-control, (former) West Berlin, Germany (51 cases and 647 controls) Jensen et hospital-based al., case-control, 198933 Copenhagen, the island of Sjaelland, Denmark (96 cases and 294 controls, identified between 1979 and 1982) crude: never used ever used 1-749 g > 750 g dose unknown Linet et al., 199535 52 m f m f m f m f m f m f m f population-based no regular use case-control, New ≤ 1.0 kg / lifetime Jersey, Iowa and Los > 1.0 kg / lifetime Angeles, US (502 cases and 496 controls identified between 1983-1986) Phenacetin adjusted for sex most cases were and exposure to included in paracetamol and previous studies tobacco adjusted for age, sex method of interview, cigarette smoking, paracetamol in any form and educational level adjusted for age this study used the and smoking same cases as McCredie et al., 1993 adjusted for socioeconomic status, cigarette smoking and laxative intake adjusted for age, sex, tobacco smoking and occupational exposures known to be associated with high risks of these cancers 79% of the tumours were located in the renal pelvis including calyces adjusted for age, sex, geographic area and cigarette smoking 308 cases with renal pelvis cancer and 194 cases with ureter cancer This study only contained small number of regular analgesic users and no analgesic abusers. Pommer et al., 199943 hospital-based and population-based case-control, West Berlin, Germany (76 cases and 647 controls) ureter cancer McCredie population-based caseet al., control, New South 198837 Wales, Australia (55 cases and 688 controls, identified between 1980-1982) renal cell cancer McLaugh population-based caselin et al., control, Minneapolis, 198541 US (495 cases and 697 controls, identified between 1974-1979) > 1.0 kg / lifetime 7/3 OR 1.8 (0.2-13) adjusted for 51 cases with renal socioeconomic pelvis and 25 cases status, cigarette with ureter cancer smoking and laxative intake. ≥ 1 kg / lifetime > 0.1 kg / lifetime 6/54 49/636 OR 1.2 (0.5-3.0) OR 0.7 (0.3-2.2) adjusted for sex and exposure to paracetamol and tobacco never 188/232 74/147 125/196 108/122 99/175 86/100 18/17 10/12 8/4 12/10 OR 1.0 OR 1.0 OR 0.7 (0.5-1.0) OR 1.7 (1.1-2.7) OR 0.7 (0.5-0.9) OR 1.7 (1.1-2.6) OR 1.3 (0.6-2.7) OR 1.9 (0.7-5.6) OR 2.2 (0.6-8.9) OR 2.4 (0.8-6.7) adjusted for age and cigarette smoking. McCredie hospital-based caseet al., control, Sidney, New 198636 South Wales, Australia (86 cases and 751 controls, identified between 1970-1982) 72/672 no consumption (lifetime exposure < 1kg) lifetime exposure > 1/35 1 kg with RPN m f m ever f m irregular f regular ≤36 mo m f regular>36 mo m f lifetime exposure > 13/44 1 kg absence of RPN McLaugh population-based case- regular use (at least 154/157 lin et al., control, Shanghai, 2 times/week for 2 198541 China (154 cases and weeks or longer) 157 controls, identified between 1978-1989) McCredie population-based caseet al., control, New South 199339 Wales, Australia (489 cases and 523 controls identified in 1989-1990) Human data non-consumers < 2.04 kg/ lifetime 2.04-6.87 kg/ lifetime > 6.88 kg/ lifetime consumption of aspirin or phenacetin adjusted for sex RR 2.5 (1.3-4.9) RR 0.4 ( 0.1-2.7) OR 2.3 (0.7-7.0) adjusted for age, sex, education, BMI and cigarette smoking. 420/474 21/16 24/16 OR 1 OR 1.4 (0.7-2.9) OR 1.8 (0.9-3.5) 17/17 OR 1.0 (0.5-2.1) adjusted for age, sex method of interview, cigarette smoking, paracetamol in any form and obesity 53 Kreiger et population-based caseal., control, Ontario, 199334 Canada (490 cases and 1351 controls, identified between 1986-1987) no phenacetin or m 265/578 acetaminophen f 166/580 phenacetin only m 2/2 f 3/7 phenacetin and m 3/4 acetaminophen f 0/8 any phenacetin m 5/6 f 3/15 McCredie case-control, data reference group m 839/1094 f 474/630 et al., pooled from studies in 199540 m 14/28 Australia, Denmark, < 0.1 kg f 17/22 Germany, Sweden and > 0.1 kg m 46/67 US (1313 cases and 1724 controls, f 51/58 identified between 0.1-1.0 kg m 25/48 1989-1991) f 26/32 1.1-5.0 kg m 16/17 f 20/14 > 5 kg m 5/2 f 5/12 Gagopopulation-based case non/irregular use 616/744 Domin- control, Los Angeles, analgesics guez et California, US (1204 regular use 86/55 al., cases and 1204 controls, max weekly dose 41/37 199932 identified between <2 g 1986-1994) max weekly dose 22/6 2-<4 g max weekly dose 23/12 4-<8 g bladder cancer McCredie population-based caseet al., control, New South 198837 Wales, Australia (162 cases and 688 controls, identified between 1980-1982) Pommer et hospital-based and al., 199943 population-based case-control, (former) West Berlin, Germany (571 cases and 647 controls, identified between 1990-1994) Castelao et population-based caseal., 200029 control, Los Angeles, USA (1514 cases and 1514 controls, 1987-1996) 54 Phenacetin OR 1.0 OR 1.0 OR 2.5 (0.3-18.5) OR 1.8 (0.5-7.3) OR 1.4 (0.3-6.7) OR 1.7 (0.5-5.9) OR 0.8 (0.2-2.7) RR 1.0 RR 1.0 RR 0.6 (0.3-1.2) RR 1.1 (0.6-2.3) RR 0.9 (0.6-1.4) RR 1.4 (0.9-2.1) RR 0.7 (0.4-1.2) RR 1.3 (0.7-2.3) RR 1.3 (0.6-2.7) RR 2.1 (1.0-4.4) RR 2.6 (0.5-14.2) RR 0.6 (0.2-1.8) OR 1.0 OR 1.9 (1.3-2.7) OR 1.3 (0.8-2.2) OR 4.1 (1.5-10.8) OR 2.3 (1.0-5.0) adjusted for age, active cigarette smoking and combined Quetelet index this study included only a small amount of phenacetin users adjusted for centre, age, sex, BMI, cigarette smoking the RR as not changed by additional adjustment for consumption of paracetamol or other analides this study only contained a small number of regular analgesics users and the amount of consumed analgesics was also small adjusted for level of education, BMI, cigarette smoking, hypertension, use amphetamines. ≥ 1 kg / lifetime 27/54 ≥ 0.1 kg / lifetime 135/636 OR 2.0 (1.1-3.5) OR 2.1 (1.3-3.5) adjusted for sex most cases were and exposure to included in previous paracetamol and studies tobacco > 1.0 kg / lifetime 23/23 OR 0.7 (0.4-1.4) adjusted for socioeconomic status, cigarette smoking and laxative intake. non/irregular use analgesics regular use < 46 g / lifetime 46-250 g / lifetime >250 g / lifetime 961/920 OR 1.0 82/64 25/18 27/20 21/20 OR 1.5 (0.9-2.7) OR 1.4 (0.6-3.1) OR 1.6 (0.7-3.7) OR 1.9 (0.8-4.4) adjusted for level of education, cigarette smoking, NSAID use, use other analgesics, employment as hairdresser Fortuny et hospital-based caseal., 200630 control, Spain (958 case and 1029 controls, identified between 1997-2000) nonusers 848/893 ever use 59/67 52/55 non regular use (> 20 times lifelong and < 2 times/week for 1 month) regular use (> 2 7/12 times/week for ≥ 1 month) Fortuny et population-based case never use 313/421 al., 200731 control, New ever use 53/35 Hampshire, UK (376 duration 4 yr 22/14 cases and 463 controls, duration 4-8 yr 6/9 identified between duration > 8 yr 25/12 1998-2001) a OR 1.0 OR 1.1 (0.7-2.0) OR 1.1 (0.6-2.0) adjusted for age, sex, region, cigarette smoking, use other NSAID or analgesics OR 1.3 (0.3-4.5) OR 1.0 OR 2.2 (1.3-3.8) OR 2.2 (1.0-4.7) OR 1.1 (0.4-3.5) OR 3.0 (1.4-6.5) adjusted for age, sex, region, cigarette smoking, use other NSAID or analgesics The number of cases and controls do not necessarily add up to the total number of cases and controls of the whole study (as mentioned in the second column), since in many studies also exposure to other (non-phenacetin-containing) analgesics are studied. Human data 55 56 Phenacetin Annex G Animal data animal species, (number, sex, age) RAT, BD I & III 30, sex unspecified, 100 d RAT, albino, 15-24, male dose, route of exposure duration carcinogenic effects ref. 40-50 mg phenacetin oral (diet) (average total dose, 22 g) 0.05, 0.1 or 0.5 % Nhydroxyphenacetin oral (diet) 0.535% phenacetin oral (diet) 2 yr no tumours observed 2 1.5 yr hepatocellular carcinomas 2 1.5-2 y carcinomas of the mammary gland and ear duct 3 RAT, S-D, 50 male, 1.25-2.5% phenacetin oral 50 female, 9 wk (diet) 1.5 yr 3 MICE, B6C3F1, 52 m+f, RAT, S-D, 20, m, 6 wk RAT, F344, 10-20, m, 6 wk 2 yr tumours in nasal cavity tumours in the urinary tract papillomas (only in female) renal cell adenoma kidney, liver, lung, skin and hemapotopoietic tumours increased labeling index kidney and bladder RAT, S-D, female RAT, Crl:CDBR 0.6-1.25 % phenacetin oral (diet) 0.05, 1.0 and 1.5% oral (diet) pretreatment 0.1% DHPN and 3.0% uracil +phenacetin 2.0% oral (diet) (1068-1145 mg/kg/d) 100, 625 and 1250 mg/kg, oral (gavage) Animal data 6-12 wk 1 50 35 wk renal cell tumours in the pre-treated rats. no tumours in the non pre-treated rats. 58 7-14 d increased DNA synthesis in respiratory and olfactory mucosa 51 57 58 Phenacetin Annex H Genotoxicity data In vitro assays. test cell line/species concentration results 0.2-2.5 mM 0.1010 mM 0.25-2.5 mM sublethal doses <10 mg/plate NT - - act. DNA fragmentation Calf thymus DNA λ DNA λ DNA gene mutation test in S.thyphimurium bacteria TA97, TA98, TA100 reverse mutation test and TA102 gene mutation test in TA98, TA 100, bacteria TA1,535, TA1,537, reverse mutation test TA1,538 DNA-repair test E.coli strains: WP2uvrA, WP67, TM1,080, TM1,080 DNA synthesis inhibition mouse L-cells test alkaline elution assay rat hepatocytes human hepatocytes unscheduled DNA liversynthesis (UDS) test hepatocytes, mouse, rat, guina pig or hamster Genotoxicity data remarks reference + act. Adams et al., 199669 NT + + TA100 De Flora et al., 198565 5,50,500, 1,000, 2,500 and 5,000 µg/plate + TA100 Oldham et al., 198666 0.3, 1, 3 mg/plate - NT De Flora et al., 198565 1 mM NT 0, 1, 1.8, 3.2 mM 0, 1, 1.8, 3.2 mM - + (rat-S9) NT NT Goto et al., 198377 Robbiano et al., 199478 0.1, 0.5, 1, 2.5, 5, 10 mM, 18-19 h - UDS measured by scintillation counting Holme et al., 198676 59 gene mutation test in mammalian cells Hprt-test V79 0, 1 and 5 mM - gene mutation test in mammalian cells Hprt-test V79 0, 1, 1.5, 5, 7.5 mM - (rat-S9) ± (hamster-S9) (rat-S9) + (hamster-S9) De Flora et al., 198565 Fassina et al., 199067 In vivo mutation assays. test species alkaline elution assay rat, liver cells rat, kidney cells route of administration i.p micronucleus test in peripheral blood cells MS/Ae mice 330 mg/kg gavage + i.p i.p i.p i.p i.p 330 mg/kg 37.5, 75, 150, 300, 400 and 600 mg/kg; 1, 2, 3 or 4 times 0, 2, 5, 50, 100 mg/kg, 1 dose 400, 600, 800 mg/kg 400, 600, 800 mg/kg 400, 600, 800 + 300, 400, 600, 800 mg/kg 400, 600 mg/kg i.p 600 mg/kg i.p micronucleus test in peripheral blood cells Sprague-Dawley rats micronucleus test in bone marrow cells in vivo gen- mutation C57BL/6 mice assay with lacZ transgenic mice 60 Phenacetin gavage oral, in feed remarks + - i.p micronucleus test in bone CD-1 mice marrow cells results gavage mouse, bone marrow i.p sister chromatid mouse i.p exchange test (SCE) micronucleus test in bone CD-1 mice i.p marrow cells micronucleus test in bone SJL Swiss mice marrow cells micronucleus test in CD-1 mice peripheral blood cells dose De Flora et al., 198565 + De Flora et al., 198565 Sutou et al., 199071 + + + + Sicardi et al., 199173 single treatment Higashikuni double treatment et al., 199274 + single treatment + double treatment + single treatment + double treatment 500, 1,000, 2,000 mg/ml during 2 days + 250, 500, 750, 1,000 mg/ml during 14 days + 250, 500, 750, 1,000 mg/ml during 14 days 0.75% w/w, 4, 8 and 12 weeks + + reference Asanami et al., 199575 sample times on day 1,3,6,9,12 and 15. sample times 4, 8 Luijten et al., or 12 weeks 200668 Annex I Carcinogenic classification of substances by the Committee The Committee expresses its conclusions in the form of standard phrases: Category Judgement of the Committee (GRGHS) 1A The compound is known to be carcinogenic to humans. • It acts by a stochastic genotoxic mechanism. • It acts by a non-stochastic genotoxic mechanism. • It acts by a non-genotoxic mechanism. • Its potential genotoxicity has been insufficiently investigated. Therefore, it is unclear whether the compound is genotoxic. The compound is presumed to be carcinogenic to humans. • It acts by a stochastic genotoxic mechanism. • It acts by a non-stochastic genotoxic mechanism. • It acts by a non-genotoxic mechanism. • Its potential genotoxicity has been insufficiently investigated. Therefore, it is unclear whether the compound is genotoxic. The compound is suspected to be carcinogenic to man. The available data are insufficient to evaluate the carcinogenic properties of the compound. The compound is probably not carcinogenic to man. 1B 2 (3) (4) Comparable with EU Category 67/548/EEC EC No 1272/2008 before as from 12/16/2008 12/16/2008 1 1A 2 1B 3 not applicable 2 not applicable not applicable not applicable Source: Health Council of the Netherlands. Guideline to the classification of carcinogenic compounds. The Hague: Health Council of the Netherlands, 2010; publication no. A10/07E.79 Carcinogenic classification of substances by the Committee 61 62 Phenacetin